Sequential exocytosis of insulin granules is associated with redistribution of SNAP25

Abstract

We have investigated sequential exocytosis in β cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in β cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-β-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

“…5c), and sometimes we observed at precisely the same site a sequence of double or triple flashes following initial fusion events, indicating longer chains of SG-SG fusions. These sequential fusion events are consistent with the 'sequential exocytosis' described previously [31]. In 3% of total fusion events, the fluorescence increase that occurred was not always associated with well-resolved sequential steps of similarly sized single SG flashes, but rather displayed massive fluorescence spots.…”

“…One mode is fusion of secretory granules, either by orderly sequential fusion as in pancreatic acinar cells [13,20] or by prior homotypic fusion leading to compound exocytosis, as in mast cells [35]. Islet beta cells employ fusion of secretory granules [31,36], but to a much lesser extent than other secretory cells. The other mode is by recruitment of newcomer secretory granules to the PM, first described in islet beta cells [11,37], but which could be a mode of exocytosis in other neuroendocrine cells.…”

“…3). Sequential SG-SG fusion and compound exocytosis have been studied in recent years, in various cell types, with multi-photon imaging [30,31] and other imaging modalities [9]. Realising that static EM images could not distinguish these exocytotic events, we employed TIRFM to rigorously and quantitatively assess the spatio-temporal dynamics of these two types of SG-SG fusion: sequential SG-SG fusion and true compound fusion.…”

Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

“…Candidates for plasma membrane factors that might diffuse into the granule membrane include RhoGEF (guanine nucleotide exchange factor for Rho) (33,34), phosphatidylinositol 4,5-bisphosphate (35), and plectin (16,36). Sequential exocytosis has also been proposed to be mediated by the lateral diffusion of apical membrane fusion proteins (10,37).…”

Métabolisme du cholestérol et function β-cellulaire

“…Ainsi la déplétion intracellulaire en cholestérol interfère avec la localisation membranaire de ces protéines. On sait que la déplétion cellulaire en cholestérol par la MCD (méthyl-b-cyclodextrine) augmente l'exocytose d'insuline dans la lignée cellulaire  HIT-15 par redistribution des canaux potassiques K v 2.1 et des SNARE en dehors des radeaux lipidiques [20], mais la littérature s'avère plutôt négative sur les effets de la MCD sur les phénomènes d'exocytose d'insuline [21][22][23]. Ces effets ne sont pas propres à la MCD.…”